In vivo Cre-recombinase Adenoviral Delivery

Isoflurane‐anesthetized C57BL/6 and mT/mG mice received 5 × 10⁸ PFU intrapleural or intraperitoneal Ad‐Cre or Ad‐Luc in 100 μl PBS and were serially imaged for bioluminescence on a Xenogen Lumina II (Perkin‐Elmer, Waltham, MA) after receiving 1 mg retro‐orbital D‐luciferin under isoflurane anesthesia, and data were analyzed using Living Image v.4.2 (Perkin‐Elmer; Stathopoulos et al, 2006 (link); Spella et al, 2019 (link)), or were euthanized and pleural lavage was performed, lungs were explanted, and parietal pleura was stripped. For pleural lavage, 1 ml PBS was injected, was withdrawn after 30 s, and was cytocentrifuged onto glass slides (5 × 10⁴ cells, 300 g, 10 min) using CellSpin (Tharmac, Marburg, Germany). Lungs were embedded in optimal cutting temperature (OCT; Sakura, Tokyo, Japan) and sectioned into 10‐µm cryosections. The parietal pleura was placed apical side up onto glass slides. Samples were stained with Hoechst 55238 and were examined on AxioObserver D1 (Zeiss, Jena, Germany) or TCS SP5 (Leica, Heidelberg, Germany) microscopes.